Proc. Fla. State Hort. Soc. 115:105-108. 2002. INVESTIGATING THE XYLELLA FASTIDIOSA IN PIERCE’S DISEASE RESISTANT AND SUSCEPTIBLE GRAPEVINES XIA XU, HONG HUANG AND JIANG LU1 Various strains of X. fastidiosa have been isolated from Florida A&M University grapevines. Some strains are virulent while others are aviru- Center for Viticulture and Small Fruit Research lent (Hopkins, 1985). The virulent strain could multiple, 6505 Mahan Drive move systemically, and reach populations of 106-107 colony- Tallahassee, FL 32317 forming units per centimeter of inoculated petioles. Aviru- lent strain of X. fastidiosa could reach only 104-105 colony- Additional index words. grape, bacterium, xylem, and sharp- forming units per centimeter and did not move beyond the shooter, Xylella fastidiosa inoculated internode (Fry and Milholland, 1990). Usually, early season infection has a long period of time for the PD Abstract. Pierce’s Disease (PD) is a major factor limiting grape bacteria to multiply, reproduce, and spread throughout the production in the southeast United States. The disease is vine. Late season infection has a short period of time for the caused by Xylella fastidiosa Wells et al., a gram-negative bac- bacteria to grow. terium that is transmitted to the xylem system of the grape- Infection of virulent strains of X. fastidiosa is fatal in some vines primarily by glassy-winged sharpshooters (Homalodisca coagulata Say). Once it is in the xylem, the X. fastidiosa bacte- grape cultivars such as most V. vinifera grapes. Infected plants rium will use the xylem sap as a nutrient source to multiply, exhibit leaf marginal scorching, cluster collapse, blackened which may eventually cause PD in susceptible cultivars. In or- and shriveled fruits, and uneven mature of stems. Dried der to understand the fate, existence, and movement of X. fas- leaves can fall off canes/stems leaving the petioles attached to tidiosa in resistant, tolerant, and susceptible grapevines the canes. Diseased vines can die in as little as one to two years during the growing and dormant seasons, the presence of X. (Hopkins, 1989). However, infection of virulent X. fastidiosa fastidiosa was investigated in field-grown and greenhouse-in- in other grape cultivars such as Florida hybrids and V. rotundi- oculated vines. X. fastidiosa was detected directly from xylem folia grapes does not appear to be a serious problem until fruit sap of field-grown grapevines by bacterium culture and con- maturation. These cultivars exhibit a certain degree of toler- firmed by polymerase chain reaction. X. fastidiosa was detect- ance or resistance to the PD infection. Infected vines are able able throughout the growing season in the PD susceptible cultivar ‘Chardonnay’ and the PD tolerant bunch grape cultivar to keep the causal agent X. fastidiosa bacterium at sub-clinical ‘Blanc du Bois’. The bacteria were also detectable in the dor- populations and appear as symptomless or recover during the mant vines with high density in these two cultivars. Xylella fas- winter (Hill and Purcell, 1997). tidiosa was found in dormant canes of muscadine grape This study was undertaken to understand the fate, exist- ‘Carlos’, but density of X. fastidiosa bacteria decreased ence, and movement of X. fastidiosa bacteria in resistant, tol- throughout the winter months. Movement of X. fastidiosa in erant, and susceptible grapevines during the growing and greenhouse-inoculated plants was indicated by the appear- dormant seasons. ance of PD symptoms on the leaves. Symptoms of PD ap- peared about five weeks after inoculation in the highly Materials and Methods susceptible cultivar ‘Chardonnay’, six weeks in ‘Niagra’, ‘Con- cord’, and the tolerant cultivar ‘Lake Emerald’, and the resis- Field-grown vines. This study was conducted at the Center tant muscadine cultivar ‘Fry’. No difference was found in PD for Viticulture and Small Fruit Research, Florida A&M Uni- sensitivity among the susceptible cultivar ‘Chardonnay’ graft- ed on different rootstocks under the greenhouse conditions. versity from 2000 to 2001 and continuing in 2002. Field- grown plants from PD tolerant muscadine grape cultivar ‘Car- los’, PD tolerant Florida hybrid bunch grape cultivar ‘Blanc Pierce’s Disease (PD) is a major factor limiting grape (Vi- du Bois’, and PD susceptible V. vinifera grape cultivar ‘Char- tis L.) production in the southeast United States. In Florida, donnay’ were used for this study. Two plants were chosen it has precluded the commercial production of European from each cultivar. Four shoots from each plant were labeled grapes V. vinifera L. and American grapes V. labrusca L. The and brought into laboratory for analysis. A Plant Moisture growth and development of a grape industry in Florida rests Stress (PMS) chamber (PMS Instrument Co., Corvallis, OR) heavily upon PD resistant or tolerant muscadine grapes V. ro- was used to extract the xylem sap unless otherwise indicated. tundifolia Michx. though incidence of PD has been reported Xylem sap was collected monthly from shoots, petioles, in several muscadine grape cultivars (Hopkins et al., 1974; Lu and roots. Bacteria were detected directly from xylem sap by et al., 2000). culturing on PD3 medium according to Hopkins (1988). A PD in grapevines is caused by Xylella fastidiosa, which is a virulent strain of X. fastidiosa bacterium, which was kindly pro- gram-negative, xylem-limited bacterium (Hopkins, 1989). X. vided by Hopkins (University of Florida, Apopka), was used as fastidiosa is transmitted into xylem of grapevines by sharp- the control. Polymerase chain reaction (PCR) was used for shooters, particularly the glassy-winged sharpshooters (Hill confirmation of X. fastidiosa. and Purcell, 1997). Once it is in the xylem, the X. fastidiosa will Symptoms of PD were rated on October 25, 2001 based on use the xylem sap as a nutrient source to multiply, spread, and a scale of 0-5 for a single plant (Fig. 1) where: 0 = no symp- increase to concentrations that clog the xylem vascular tissue toms; 1 = minor symptoms up to 10% of leaves with marginal and can eventually cause PD in susceptible grape cultivars. necrosis; 2 = 11-30% of leaves with marginal necrosis; 3 = 31- 50% of leaves with marginal necrosis; 4 = 51-75% of leaves 1Corresponding author. with marginal necrosis, or dead growing point and; 5 = over Proc. Fla. State Hort. Soc. 115: 2002. 105 Fig. 1. Pierce’s Disease (PD) symptoms. 75% of leaves with marginal necrosis or a dead arm. The same method was used for evaluating PD severity of single shoots. Ratings of PD for a single leaf on a shoot were based on a 0-4 scale where: 0 = no symptoms; 1 = 0-25% of leaf area with mar- ginal necrosis; 2 = 26-50% of leaf area with marginal necrosis; 3 = 51-75% of leaf area with marginal necrosis and; 4 = over 75% of leaf area with marginal necrosis. Greenhouse-grown vine. PD susceptible V. vinifera grape ‘Chardonnay’ and V. labrusca grapes ‘Niagra’, and ‘Concord’, PD tolerant Florida hybrid bunch grape cultivar ‘Lake Emer- ald’, and PD resistant muscadine grape cultivar ‘Fry’ were Fig. 2. Diagram of inoculation position of X. fastidiosa in a greenhouse- used in greenhouse study. Five plants from each cultivar were grown plant. used. Three of them were inoculated on 5 May 2001 with sus- pension culture of X. fastidiosa by using needle puncture tech- nique (Hopkins, 1989). Two vines were used as controls ceptible ‘Chardonnay’, only shoots with PD ratings over 2 had which were inoculated with PD3 medium only. Each plant was bacteria growth on PD3 medium. There were relatively less inoculated in section A—internode between 2nd and 3rd bacterial colonies on the culture of xylem sap from ‘Blanc du leaves from the bottom of the plant (Fig. 2). This operation Bois’ than from ‘Chardonnay’. In the PD tolerant muscadine was repeated three times every 6 days in the greenhouse of ‘Carlos’, bacteria were also isolated from shoots with PD symp- Florida A&M University using a completely random design. A toms during most months of 2001 and the early part of 2002. similar experiment was conducted to investigate the root- However, the number of bacterial colonies in ‘Carlos’ was sig- stock effect on PD occurrence in V. vinifera cultivar ‘Chardon- nificantly less than in ‘Chardonnay’ and ‘Blanc du Bois’. nay’ grafted on different rootstocks. PCR confirmation of Xylella fastidiosa bacteria. Although bacte- Symptoms of PD appearance were recorded every week in ria were detectable in all tested grape cultivars by medium cul- three sections: section A, as described above; section B being ture, not all of them were X. fastidiosa bacteria when they were the internode between the 4th and 5th leaves, and; section C used for amplification of the specific DNA sequence of X. fas- being the internode between the 6th and 7th leaves. The rest tidiosa bacteria by using polymerase chain reaction (Table 1). of the leaves have marked as others. Observation of PD began X. fastidiosa bacteria were consistently confirmed from the PD3 one week after inoculation and continued every week for five medium cultures in PD susceptible ‘Chardonnay’ throughout months. the year of 2001. However, X. fastidiosa bacteria were detect- able in PD tolerant ‘Blanc du Bois’ only in January, February, Results and Discussions August, September, October, November, and December of the year 2001. X. fastidiosa bacteria could not be detected in Isolation of bacteria from field-grown vines. The appearance of those vines from March to July 2001. Similar results were also bacteria in xylem sap during the growing and dormant sea- obtained during the first half of 2002. For the PD tolerant mus- sons varied among the resistant, tolerant, and susceptible cadine ‘Carlos’, X. fastidiosa was not detectable until Septem- grapevines when cultured on PD3 medium (Table 1). In the ber, 2001. Positive PCR was continually confirmed for the highly PD susceptible European cultivar ‘Chardonnay’, bacte- following four months until January, 2002.